General regulatory factors exert differential effects on nucleosome sliding activity of the ISW1a complex

Background Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown. Results Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome. Conclusions Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly. Supplementary Information The online version contains supplementary material available at 10.1186/s40659-024-00500-6.

The image shows a Coomassie bluestained gel (12%) of His-tagged proteins expressed in and purified from E. coli BL-21.The identity of each purified protein is depicted at the top of the gel picture.MW: molecular weight marker (Unstained Protein Standard, Broad Range, New England Biolabs P7717S).BSA: bovine serum albumin (BioRad 5000206), used for densitometric quantification of the recombinant proteins.The oval represents the translational position adopted by the nucleosome core upon reconstitution, which covers the 601 region.The term "NC" in probe names stands for "nucleosome core".The probe name indicates length of linker DNA upstream (left) and downstream (right) of the NC and presence of the Rap1 binding site (Rap1bs).(B) Outline of the steps involved in the assays.The Removing mix consist in a mix that contains 500 ng of an unrelated non-labeled DNA and a non-labeled double-stranded oligonucleotide harboring the Rap1's target sequence, added in an excess of 100x, 200x or 300x relative to Rap1 concentration (100x in figure C). (C) EMSA testing the effect of temperature on the efficiency of Rap1 removal from its target sequence.The image corresponds to electrophoresis in a non-denaturing polyacrylamide gel.Each temperature was tested in duplicate.The conditions for each reaction are depicted at the top of the gel picture, as well as percentages of bound probe; migration of the nucleosome probe is indicated schematically at the right of the gel picture, as well as migrations of free DNA probe (DNA), and nucleosome probe bound by Rap1 (Rap1-Nuc).(D) EMSA testing effect of increasing amounts of competitor oligonucleotide on the efficiency of Rap1 removal from its target sequence in the probe.Removal incubations were performed at 37°C in all reactions of this assay.The image corresponds to electrophoresis in a nondenaturing polyacrylamide gel.The conditions for each reaction are depicted at the top of the gel picture, as well as percentages of bound probe; migration of the nucleosome probe is indicated schematically at the right of the gel picture, as well as migrations of free DNA probe (DNA), and nucleosome probe bound by Rap1 (Rap1-Nuc).

Figure S3. High nucleosome occupancy and histone deposition levels are mainly present at loci displaying low affinity or low occupancy levels of Rap1. (A)
Scatter plots displaying nucleosome occupancy (left panel) and histone deposition (right panel) levels at loci bound by Rap1, relative to affinity levels of Rap1 at these loci.Nucleosome occupancy and histone deposition levels were determined from genome-wide ChIP-seq data obtained in the study performed by Kassem and co-workers (1).An analysis of protein binding to purified genomic DNA coupled to deep sequencing (PB-exo), performed by Rossi and co-workers (2), was used to define affinities of Rap1 to its target sequences genome-wide.(B) Scatter plots displaying nucleosome occupancy (left panel) and histone deposition (right panel) levels at loci bound in vivo by Rap1, relative to occupancy levels of Rap1 at these loci.Nucleosome occupancy and histone deposition levels were determined from genome-wide ChIP-seq data obtained in the study performed by Kassem and co-workers (1).The in vivo genome-wide occupancy levels of Rap1 were obtained from a ChIP-exo analysis performed by Rossi and co-workers (2).
Table S1.Sequence information of template plasmid and primers used for generation of each probe.Sequences highlighted in yellow correspond to the regions bound by the PCR primers.

Figure S1 .
Figure S1.SDS-PAGE analysis of purified His-tagged transcription factors.The image shows a Coomassie bluestained gel (12%) of His-tagged proteins expressed in and purified from E. coli BL-21.The identity of each purified protein is depicted at the top of the gel picture.MW: molecular weight marker (Unstained Protein Standard, Broad Range, New England Biolabs P7717S).BSA: bovine serum albumin (BioRad 5000206), used for densitometric quantification of the recombinant proteins.

Figure S2 .
Figure S2.Standardization of Rap1 removal for nucleosome remodeling assays (A) Schematic representation of the nucleosome probe used in the standardization assays.601 NPS = nucleosome positioning region of the 601 sequence (gray bar; 147 bp).The oval represents the translational position adopted by the nucleosome core upon reconstitution, which covers the 601 region.The term "NC" in probe names stands for "nucleosome core".The probe name indicates length of linker DNA upstream (left) and downstream (right) of the NC and presence of the Rap1 binding site (Rap1bs).(B) Outline of the steps involved in the assays.The Removing mix consist in a mix that contains 500 ng of an unrelated non-labeled DNA and a non-labeled double-stranded oligonucleotide harboring the Rap1's target sequence, added in an excess of 100x, 200x or 300x relative to Rap1 concentration (100x in figureC).(C) EMSA testing the effect of temperature on the efficiency of Rap1 removal from its target sequence.The image corresponds to electrophoresis in a non-denaturing polyacrylamide gel.Each temperature was tested in duplicate.The conditions for each reaction are depicted at the top of the gel picture, as well as percentages of bound probe; migration of the nucleosome probe is indicated schematically at the right of the gel picture, as well as migrations of free DNA probe (DNA), and nucleosome probe bound by Rap1 (Rap1-Nuc).(D) EMSA testing effect of increasing amounts of competitor oligonucleotide on the efficiency of Rap1 removal from its target sequence in the probe.Removal incubations were performed at 37°C in all reactions of this assay.The image corresponds to electrophoresis in a nondenaturing polyacrylamide gel.The conditions for each reaction are depicted at the top of the gel picture, as well as percentages of bound probe; migration of the nucleosome probe is indicated schematically at the right of the gel picture, as well as migrations of free DNA probe (DNA), and nucleosome probe bound by Rap1 (Rap1-Nuc).
Letters in red correspond to the nucleosome positioning region of Widom's 601 sequence(3).